Use the following steps to create a custom Gateway vector to use in SeqBuilder:
1. Open your vector sequence file and make sure it is set to circular by selecting Sequence>Circular.
2. Add misc_recomb features to your sequence on the appropriate sub-sequences where recombination occurs. Note that while it is preferred for the entire recombination site to exist within your sequence, SeqBuilder at minimum requires the appropriate att core sequence for each of the five types of recombination sites. SeqBuilder uses these core sequences along with the feature labels to correctly identify each Gateway plasmid:
• Type 1: GTACAAA (top strand)
• Type 2: CTTGTAC (bottom strand)
• Type 3: ATTATAC (bottom strand)
• Type 4: GTATAGA (top strand)
• Type 5: GTATACA (top strand)
3. Label the features as follows:
• For Expression Clones: attB1, attB2, attB3, attB4, and attB5
• For Donor Vectors: attP1, attP2, attP3, attP4, and attP5
• For Entry Clones: attL1, attL2, attL3, attL4, and attL5
• For Destination Vectors: attR1, attR2, attR3, attR4, and attR5
Note: The classic Gateway cloning protocol uses only types 1 and 2 of recombination sites. MultiSite Gateway technology utilizes all 5 types of recombination sites, which allows you to recombine multiple regions at one time.
4. Save your sequence file.
Once this is done, you can add your custom vector to the catalog so that it can be used in all projects:
1. Select File>Open.
2. Select the vectors catalog, CloningVectors.sbp,found in the following directory:
Windows XP: C:\Documents and Settings\All Users\Shared Documents\DNASTAR\Lasergene 'x' Data
Windows Vista, Windows 7 and Windows 8: C:\Users\Public\Public Documents\DNASTAR\Lasergene 'x' Data
Macintosh OSX: Hard Drive:Applications:DNASTAR:Lasergene 'x' Data
...where 'x' represents the version number.
3. Expand the Gateway Vectors folder, then select the appropriate sub-folder, depending on the type of custom vector you are adding.
4. File > Import Sequence(s) to Project and navigate to your custom vector sequence file. Selecting this sequence file will add it to the vector folder you selected.